A central feature of the plasma membrane (PM) of eukaryotic cells is the asymmetric distribution of various lipid types across the two membrane leaflets. Maintaining such transbilayer organization requires investment of significant energetic resources, implying an important functional role for interleaflet lipid segregation. A prominent example are charged lipids like phosphatidylserine (PS): normally restricted to the cytoplasmic PM leaflet in healthy cells, their permanent exposure on the exoplasmic face marks the cell for phagocytosis by macrophages. The paradigm has recently been challenged by observations that PS temporarily flips to the outer PM leaflet during antigen-induced activation in healthy immune cells. The purpose and mechanisms of this phenomenon are wholly unknown, nor is it clear whether this redistribution is specific for PS, or rather leads to wholesale lipidomic and biophysical scrambling of the two PM leaflets. To investigate these intriguing questions, we propose to study the characteristics, regulatory mechanisms, and functional roles of this reversible lipid scrambling during immune cell activation. In Aim 1, we will use advanced lipidomics and imaging techniques to characterize the changes in membrane lipid organization during immune cell activation. We hypothesize that antigen activation induces transient, highly localized, and non-specific lipid scrambling, in contrast to the canonical permanent PS exposure associated with apoptosis. In Aim 2, we will identify the molecular mechanisms responsible for this effect. Using super-resolution microscopy, we will monitor the nanoscale organization of various lipid translocators, flipped lipids, and key machinery in the immune signaling cascade. We expect to observe spatial segregation and confinement of these key molecules into specialized PM domains. Finally, in Aim 3 we will examine the functional implications of lipid scrambling by evaluating the effect of PM asymmetry on protein-membrane interactions. We hypothesize that activation-induced scrambling reduces the charge density of the inner PM leaflet, leading to dissociation of charge-dependent signaling proteins from the membrane. To test this hypothesis, we will use fluorescence microscopy in situ?as well as biophysical tools in vitro and in silico?to examine the membrane binding affinity of polybasic-domain containing proteins, including the oncogene Ras, in symmetric and asymmetric membranes. The successful execution of these aims will produce important and novel insights into physiologically regulated changes in membrane organization and its role in immune cell activation. Moreover, these studies will provide the first detailed lipidomic and biophysical characterization of both unperturbed and functionally modified PM asymmetry. The findings may be extensible to cell activation in a variety of other contexts, and are thus expected to have far-reaching impacts by revealing new potential targeting for understanding and treating pathologies.